本文選題：代謝綜合征 + RCT��； 參考：《北京協(xié)和醫(yī)學(xué)院》2015年博士論文

【摘要】：代謝綜合征是指人體內(nèi)的蛋白質(zhì)、脂肪、碳水化合物等物質(zhì)發(fā)生代謝紊亂,從而造成的一組復(fù)雜的代謝紊亂癥候群,是導(dǎo)致糖尿病、心腦血管疾病等的危險(xiǎn)因素。其中心腦血管疾病是當(dāng)今世界范圍內(nèi)發(fā)病率、致死率最高的重大疾病之一,動(dòng)脈粥樣硬化(Atherosclerosis, AS)是其主要的病理基礎(chǔ),而脂質(zhì)代謝尤其是膽固醇代謝紊亂又是AS的主要致病因素。早期抗動(dòng)脈粥樣硬化藥物的研究以調(diào)脂為主要特征,他汀類藥物是典型代表。這類藥物雖然可以有效地降低膽固醇,但是其總的有效率僅為30-40%。糖尿病(DM, diabetes mellitus)是代謝綜合征中一個(gè)非常重要的危險(xiǎn)因素,現(xiàn)已成為第三大危害人類健康的疾病。過氧化物酶體增殖物激活受體γ(Peroxisome Proliferator-Activated Receptor y, PPARy)在調(diào)節(jié)糖脂代謝穩(wěn)態(tài)上具有重要的作用,是目前臨床使用的噻唑烷二酮(Thiazolidinediones, TZDs)類藥物的分子靶點(diǎn)。然而,TZDs完全激活PPARγ可產(chǎn)生一系列不期望的副作用,如體重增加、水腫和組織損傷等。因此,開發(fā)兼能調(diào)節(jié)糖代謝和脂代謝的新型藥物極具前景。近年來,脂代謝過程中的關(guān)鍵途徑——膽固醇逆向轉(zhuǎn)運(yùn)(reverse cholesterol transport,RCT)通路及選擇性PPARγ調(diào)節(jié)劑(Selective PPARγ Modulator, SPPARyM)受到了越來越多的關(guān)注。鑒于此,我們進(jìn)行了靶向RCT的先導(dǎo)化合物的篩選、藥理學(xué)、機(jī)制研究以及PPARγ選擇性激動(dòng)劑虛擬篩選模型的構(gòu)建與應(yīng)用研究：1.靶向RCT的先導(dǎo)化合物改善脂質(zhì)和葡萄糖代謝的藥理學(xué)及機(jī)制研究RCT是指膽固醇由外周組織轉(zhuǎn)運(yùn)回肝臟進(jìn)行再循環(huán)或以膽汁酸的形式隨糞便排出體外的過程。ABCA1、ABCG1是ATP結(jié)合盒轉(zhuǎn)運(yùn)體超家族成員,介導(dǎo)細(xì)胞內(nèi)磷脂和游離膽固醇轉(zhuǎn)運(yùn)到貧脂或無脂的ApoA-I,從而促進(jìn)HDL的生成,啟動(dòng)RCT過程。SRB1為功能性的高密度脂蛋白受體。這些蛋白都是調(diào)控RCT過程的關(guān)鍵因子。本論文的第一部分工作是以實(shí)驗(yàn)室前期構(gòu)建的ABCA1/SRB1表達(dá)上調(diào)劑篩選模型為基礎(chǔ),對國家新藥(微生物)篩選實(shí)驗(yàn)室的化合物庫進(jìn)行篩選,得到了活性化合物E17241和E22840,它們能夠在體外分子水平調(diào)節(jié)參與膽固醇代謝、轉(zhuǎn)運(yùn)、分泌以及脂肪酸代謝的蛋白表達(dá),并且能夠有效地促進(jìn)巨噬細(xì)胞中的膽固醇外排,減少巨噬細(xì)胞泡沫化的發(fā)生,減少細(xì)胞內(nèi)脂質(zhì)堆積。在動(dòng)脈粥樣硬化模型ApoE-/-、鼠體內(nèi),E17241和E22840均能夠降低血漿中膽固醇、甘油三酯以及LDL的水平,增加HDL的含量,同時(shí)主動(dòng)脈和心臟流出道中的動(dòng)脈粥樣硬化斑塊也明顯減少。另外,小鼠肝臟內(nèi)的脂質(zhì)含量也明顯下降,通過對肝臟細(xì)胞中的相關(guān)蛋白表達(dá)的檢測說明E17241和E22840是通過調(diào)節(jié)ABCA1、ABCG1、SRB1等蛋白來將膽固醇逆轉(zhuǎn)運(yùn)至肝臟中進(jìn)行代謝。除此之外,我們還發(fā)現(xiàn)這兩個(gè)活性化合物能夠激活PPARy,其中E17241不僅能夠顯著降低ApoE-/-小鼠的血糖濃度,還能夠降低2型糖尿病模型KKAy小鼠的空腹血糖及胰島素水平,改善其葡萄糖耐受能力和胰島素敏感性。不僅如此,在KKAy小鼠體內(nèi),E17241還能夠緩解其由于肥胖造成的高血脂,減少肝臟中的膽固醇含量。相對于羅格列酮,它對肝、腎的損傷較小。另外,我們還發(fā)現(xiàn)該化合物之所以能夠上調(diào)ABCA1和SRB1的表達(dá),部分原因可能是其通過PKC/PKA通路激活了PPARs和LXRs等核受體,并且增加了SRB1蛋白的穩(wěn)定性�；衔顴17241和E22840的活性均為首次報(bào)道,可作為靶向RCT的先導(dǎo)化合物,在體內(nèi)外發(fā)揮對膽固醇代謝和葡萄糖代謝的調(diào)節(jié)作用,具有進(jìn)一步開發(fā)成為抗動(dòng)脈粥樣硬化藥物的潛能,其中E17241還有望發(fā)展成為治療糖尿病并同時(shí)緩解其伴隨的高血脂和脂肪肝的新型候選藥物。2.PPAR丫選擇性激動(dòng)劑的虛擬篩選及藥理學(xué)研究PPARy是配體結(jié)合的核受體超家族中的一員,主要分布在白色和棕色脂肪,它在調(diào)節(jié)脂肪細(xì)胞分化、脂質(zhì)代謝、維持葡萄糖體內(nèi)平衡和胰島素敏感性中發(fā)揮重要作用。完全激活PPARy可誘導(dǎo)多組織非特異性基因的表達(dá),而SPPARγM則能夠選擇性地調(diào)控某些特定基因的轉(zhuǎn)錄。目前有許多具有PPARγ部分激動(dòng)活性的化合物在臨床前試驗(yàn)中表現(xiàn)出與完全激動(dòng)劑相當(dāng)?shù)囊葝u素增敏作用,但是具有較弱的或基本不產(chǎn)生TZDs的副作用。本論文的第二部分工作主要是尋找和發(fā)現(xiàn)新型的PPARy選擇性激動(dòng)劑。我們建立了基于藥效團(tuán)的虛擬篩選工作流程,對國家新藥(微生物)篩選實(shí)驗(yàn)室的化合物庫進(jìn)行了40,000樣次篩選,獲得了活性化合物F12016和F12025。這兩個(gè)化合物是對PPARγ亞型具有選擇性,激動(dòng)PPARγ的最大效能也只有羅格列酮的20-30%,并且能夠拮抗羅格列酮的激動(dòng)作用。此外,F12016和F12025能夠促進(jìn)肝細(xì)胞和脂肪細(xì)胞代謝葡萄糖,同時(shí)明顯減少了對脂肪細(xì)胞分化的誘導(dǎo)。它們由于與PPARγ相互作用的方式與羅格列酮不同,因此會(huì)產(chǎn)生不同的構(gòu)象改變,并募集特定的輔因子,從而不同程度的調(diào)節(jié)靶基因的表達(dá)。在白發(fā)性2型糖尿病模型KKAy小鼠體內(nèi),F12016能夠明顯降低小鼠的空腹血糖,改善葡萄糖耐受性和胰島素敏感性,但不會(huì)引起明顯的體重增加。與羅格列酮相比,F12016還能夠明顯減少對成骨細(xì)胞分化的抑制作用。我們還研究了F12016和F12025與PPARy的相互作用模式,并初步探討了其既能保留藥理學(xué)作用又能減少副作用的原因可能是由于其相當(dāng)程度的抑制了CDK5介導(dǎo)的PPARγ Ser273磷酸化。因此,化合物F12016和F12025可作為PPARγ選擇性激動(dòng)劑,有可能發(fā)展成為新型抗糖尿病的先導(dǎo)化合物。綜上,本論文獲得了靶向RCT的先導(dǎo)化合物E17241和E22840,在分子、細(xì)胞水平確證了其生物學(xué)活性并進(jìn)行了體內(nèi)藥理學(xué)研究；構(gòu)建了適用于高通量篩選PPARγ選擇性激活劑的虛擬篩選模型,通過篩選得到了活性較好的F12016和F12025,確證了其體內(nèi)外生物學(xué)活性并初步探討了其機(jī)制。本論文工作為尋找新型抗代謝綜合征藥物奠定了基礎(chǔ),具有重要的理論和實(shí)踐意義。
[Abstract]:Metabolic syndrome is a complex metabolic disorder syndrome caused by metabolic disorders in the body, such as protein, fat, carbohydrate, etc., which is a risk factor for diabetes, cardiovascular and cerebrovascular diseases, among which cardiovascular and cerebrovascular diseases are one of the most fatal diseases in the world. Atherosclerosis (AS) is the main pathological basis, and lipid metabolism, especially cholesterol metabolism disorder, is the main pathogenic factor of AS. Early anti atherosclerotic drugs are mainly characterized by lipid regulation, statins are typical. Although these drugs can effectively reduce cholesterol, but their total The effective 30-40%. diabetes (DM, diabetes mellitus) is a very important risk factor in the metabolic syndrome. It has now become the third major health hazard. Peroxisome proliferator activated receptor gamma (Peroxisome Proliferator-Activated Receptor y, PPARy) is important in regulating the homeostasis of glycolipid metabolism. The role is the molecular target of Thiazolidinediones (Thiazolidinediones, TZDs), which is currently used clinically. However, the complete activation of PPAR gamma by TZDs can produce a series of undesirable side effects, such as weight gain, edema and tissue damage. Therefore, the development of new drugs which can regulate glucose metabolism and lipid metabolism is very promising. The key pathways in metabolic processes - reverse cholesterol transport (RCT) pathway and selective PPAR gamma modulator (Selective PPAR gamma Modulator, SPPARyM) have attracted more and more attention. In view of this, we have screened the precursor compounds for targeting RCT, pharmacology, mechanism and PPAR gamma selectivity. Study on the construction and application of a virtual screening model for agonists: 1. the pharmacological and Mechanism Research on the improvement of lipid and glucose metabolism by the pilot compound targeting RCT RCT refers to the transfer of cholesterol from peripheral tissue to the liver for recirculation or in the form of bile acid in the form of fecal excretion of.ABCA1, ABCG1 is a ATP binding cassette transporter. Members of the family, which mediate intracellular phospholipids and free cholesterol transport to poor fat or fat free ApoA-I, thus promote the generation of HDL and start the RCT process as a functional high-density lipoprotein receptor. These proteins are the key factors in the regulation of RCT processes. The first part of this paper is to increase the expression of ABCA1/SRB1 in the early laboratory. On the basis of the agent screening model, the compound library of the national new drug (microorganism) screening laboratory is screened and the active compounds E17241 and E22840 are obtained. They can regulate the protein table of cholesterol metabolism, transport, secretion and fatty acid metabolism at the molecular level in vitro, and can effectively promote the cholesterol in macrophages. In the atherosclerotic model ApoE-/-, E17241 and E22840 can reduce the levels of cholesterol, triglycerides and LDL in plasma, increase the content of HDL, and also decrease the atherosclerotic plaques in the aorta and the heart outflow tract. In addition, the lipid content in the liver of mice also decreased significantly. By detecting the expression of related proteins in the liver cells, E17241 and E22840 were metabolized by reversing cholesterol by regulating ABCA1, ABCG1, SRB1 and other proteins. In addition, we also found that these two active compounds can activate PPARy, of which E17241 is not. It can only significantly reduce the blood glucose concentration in ApoE-/- mice and reduce the fasting blood glucose and insulin level in type 2 diabetes model KKAy mice, improve their glucose tolerance and insulin sensitivity. Not only so, in KKAy mice, E17241 can also alleviate hyperlipidemia caused by fat fat and reduce the cholesterol content in the liver. Relative to rosiglitazone, it has less damage to the liver and kidney. In addition, we also found that the compound can increase the expression of ABCA1 and SRB1, partly due to the activation of PPARs and LXRs nuclear receptors through the PKC/PKA pathway, and the increase of the stability of SRB1 protein. The activity of compound E17241 and E22840 is reported for the first time. As a pilot compound for targeting RCT, it plays a role in regulating cholesterol metabolism and glucose metabolism in vivo and in vivo and has the potential to be further developed as an anti atherosclerotic drug, and E17241 is also expected to develop into a new candidate drug,.2.PPAR, for the treatment of diabetes and the simultaneous release of hyperlipidemia and fatty liver. The virtual screening and pharmacological study of selective agonists PPARy is a member of the ligand binding nuclear receptor superfamily, mainly distributed in white and brown fat. It plays a vital role in regulating adipocyte differentiation, lipid metabolism, maintaining glucose homeostasis and insulin sensitivity. Fully activating PPARy can induce multi tissue nonspecific. The expression of the heterosexual genes, while SPPAR gamma M can selectively regulate the transcription of certain specific genes. Many of the compounds with PPAR gamma partial excitant activity present in preclinical trials show the insulin sensitization equivalent to complete agonists, but there are weak or basic side effects that do not produce TZDs. Second Part of the work is to find and discover new PPARy selective agonists. We set up a virtual screening workflow based on the pharmacophore, and screened the compound library of the national new drug (microorganism) screening laboratory for 40000 times. The two compounds, active compounds and F12025., were selected for the selection of PPAR gamma subtypes. The maximum efficiency of PPAR gamma is only the 20-30% of rosiglitazone, and it can antagonize the agitation of rosiglitazone. In addition, F12016 and F12025 can promote the metabolism of glucose in liver and fat cells, and obviously reduce the induction of adipocyte differentiation. They are different from rosiglitazone in the way of interaction with PPAR gamma. Therefore, different conformation changes will be produced and a specific cofactor is raised to regulate the expression of the target gene in varying degrees. In the white hair type 2 diabetes model KKAy mice, F12016 can significantly reduce the fasting blood glucose in mice, improve glucose tolerance and insulin sensitivity, but do not cause significant weight gain. F12016 can also significantly reduce the inhibitory effect on osteoblast differentiation. We also studied the interaction patterns of F12016 and F12025 and PPARy, and preliminarily discussed the reason that it could retain the pharmacological action and reduce the side effects because of its considerable inhibition of the CDK5 mediated phosphorylation of PPAR gamma Ser273. Therefore, compounds F12016 and F12025 can be used as PPAR gamma selective agonists and may develop into a new type of antidiabetic precursor compound. In this paper, E17241 and E22840, a precursor of target RCT, were obtained in this paper, and their biological activities were confirmed at the molecular and cell levels. The virtual screening model of PPAR gamma selective activator was screened and the active F12016 and F12025 were obtained by screening. The biological activity of the active agent was confirmed and its mechanism was preliminarily discussed. This work has laid the foundation for finding new anti metabolic syndrome drugs, which has important theoretical and practical significance.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R589

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 Teayoun Kim;Qinglin Yang;;Peroxisome-proliferator-activated receptors regulate redox signaling in the cardiovascular system[J];World Journal of Cardiology;2013年06期

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本文編號：2116511